Glycogen synthase kinase-3 (GSK-3), a serine/threonine kinase, plays an important role in regulating growth and death of cardiomyocytes. During the current funding cycle, we established that GSK-3 is a negative regulator of cardiac hypertrophy and has substantial influence on cardiac function when its activity is modulated by phosphorylation during hypertrophy and heart failure (HF). Furthermore, our recent study conducted using GSK-3a(S21A) and GSK-3b(S9A) knock-in (KI) mice demonstrated that GSK-3 has isoform specific functions, which may be attributed to the distinct subcellular localizations of GSK-3a and GSK-3b. Unexpectedly, S21 phosphorylation of GSK-3a and S9 phosphorylation GSK-3b exhibited opposite functional consequences in the heart under pressure overload, indicating the importance of re-evaluating the function of the GSK-3 isoforms with special emphasis on their subcellular localization and unique targets. Thus, one important theme in this proposal is to demonstrate that endogenous GSK-3a and GSK-3b, localized in different subcellular locations/compartments, play distinct roles in mediating growth, death and differentiation of cardiomyocytes and their precursor cells. In particular, we will elucidate the isoform-specific functions of GSK-3 during pressure overload-induced cardiac hypertrophy, ischemia/reperfusion (I/R) and differentiation of bone marrow (BM)-derived mesenchymal stem cells (MSCs) by focusing on novel connections between GSK-3 isoforms and their downstream targets. Our hypotheses are: 1. GSK-3a, primarily localized in the nucleus, regulates expression of E2F in adult hearts. Phosphorylation of GSK-3a and subsequent upregulation of E2F is a compensatory mechanism to supplement myocyte proliferation and prevent mitochondrial dysfunction during pressure overload. 2. GSK-3b regulates survival and death of cardiomyocytes during I/R. Activation of GSK-3b during ischemia inhibits mTOR through a TSC2-dependent mechanism, stimulates autophagy, and protects the heart from cell death. On the other hand, phosphorylation/inactivation of GSK-3b during reperfusion is beneficial through activation of mTOR. 3. Upregulation of GSK-3b and downregulation of GSK- 3a facilitate differentiation of BM-derived MSCs into the cardiomyocyte lineage through distinct molecular mechanisms. Injection of MSCs in which GSK-3b is upregulated and GSK-3a is downregulated ex vivo facilitates the recovery of the heart after myocardial infarction (MI) through stimulation of cardiomyocyte differentiation and angiogenesis. We will address these issues, using genetically altered mouse models and integrated molecular and physiological approaches. Our study will elucidate the isoform-specific functions and unique downstream targets of GSK-3a and GSK-3b in mediating both growth/death and differentiation in the heart under stresses. The knowledge obtained from this investigation will lead to a better understanding of the molecular mechanisms mediating HF, ischemic injury, and stem cell differentiation, which can be utilized to develop specific interventions to treat HF and ischemic heart disease, and improve cell-based therapies.